(a) Field of the Invention
The invention relates to kits for the accurate, rapid and sensitive assay of A. pleuropneumoniae serotype 2 antibodies in pig serum for swine pleuropneumonia serodiagnosis.
(b) Description of Prior Art
Actinobacillus pleuropneumoniae is known as one of the most pathogenic agents of the respiratory tract of swine. Swine pleuropneumonia is still an important problem in large swine operations, causing serious economic losses in this industry. Since the presence of A. pleuropneumoniae is often unnoticed in chronically infected herds, the identification of carrier animals is a main concern. Following a stressful situation, several clinically fatal cases may arise in a given herd. Infection in swine can be fatal but animals surviving the infection frequently become carriers. Detection of chronically infected carriers is crucial since those animals act as reservoirs of infection. Since the infection is often unnoticed, serology becomes a useful tool for the detection of chronic infection. Several studies indicate that it is possible to control or eliminate the infection in certain herds based on the serological results.
Various serological assays for A. pleuropneumoniae have been described. Among others, the complement fixation test (CFT), the enzyme-linked immunosorbent assay (ELISA); (Goyette G. et al., 1986, Int. Pig. Vet. Soc. Proc., 9:258) and the 2-mercapto-ethanol tube agglutination test (Mittal, K. et al., 1984, Am.J. Vet.Res., 45:715-719) have been used. Out of the different assays, the ELISA is often the most useful since it is faster and easier to perform. On the other hand, up to now, the results obtained suggested the use of a more purified antigenic preparation in order to improve the specificity of the test.
A saline extract of boiled-formalinized whole cells of A. pleuropneumoniae (also called crude extract) is presently used, in some laboratories, as the antigen for ELISA serodiagnosis (Goyette G. et al., 1986, Int. Pig. Vet. Soc. Proc., 9:258). Standardization of the assay is complicated as variations are noticed between extracts.
Using different antigen preparations, cross-reactions among serotypes and with other bacterial species were also reported (Bossxc3xa9, J. et al., 1990, Can. J. Vet. Res., 54:427-431). Although the capsular polysaccharide (CPS) of A. pleuropneumoniae has been shown to be responsible for serotype specificity (Inzana, T. and Mathison, T., 1987, Infect. Immun., 55:1580-1587), the difficulty of obtaining pure CPS in large quantity precludes its utilization for serodiagnostic purposes. The CPS were very unstable and were fixed with difficulty to the walls of the polystyrene plate used in the ELISA assay (Perry, B. et al., 1990, Sero. Immunol. Infect. Dis., 4:299-308).
Serology, which is used to identify animals that have developed an immune response to specific pathogens, is an important tool in disease management and prevention of A. pleuropneumoniae infection in pigs. The importance of serological testing is further emphasized by the lack of a vaccine that reliably prevents infection.
The use of antibiotics is mainly useful to control the mortality, but it has no real benefit on pigs with chronic pleuropneumonia. Treated animals often continue to carry the organism and can be a source of infection for other animals. In addition, an increasing number of strains resistant to different antimicrobials has been observed in the last years in Quebec (Nadeau, M. and Higgins, R., 1991, Bulletin xc3xa9pidxc3xa9miologique, 2:4-5).
The demand of pigs from A. pleuropneumoniae sero-negative herds is increasing, especially from producers whose herds have experienced acute outbreaks of the disease and who have decided to xe2x80x9ceradicatexe2x80x9d A. pleuropneumoniae, buying only sero-negative animals (coming from sero-negative herds) for the replacement. A successful eradication program depends mostly on the accuracy and reliability of the serological tests used to identify A. pleuropneumoniae infected pigs. Never-theless, interpretation of serology should be done cautiously. A test that is not sensitive will not detect all infected herds or animals (false negative results) and one that is not specific will erroneously condemn some non-infected animals (false positive results).
The antigenic specificity of A. pleuropneumoniae appears to be linked, at least partly, to the capsular polysaccharides (Altman et al., 1988, Biochem. Cell. Biol. 66:998-1004; Benyon et al., 1991, Carbohydrate Res., 209:211-223; Bosse et al., 1990a, Can. J. Res. 54:320-325, and 1990b, Can. J. Vet. Res. 54:427-431) or, according to other authors, to the smooth lipolysaccharides (Altman et al., 1989, Carbohydrate Res. 191:295-303; Benyon et al., 1991, Carbohydrate Res. 209:225-238). However, these capsular polysaccharides turn out to be very unstable and are difficult to attach to the polystyrene surfaces of the plates used for the ELISA (Perry et al., 1990, Immunother. Infect. Dis. 4:299-308; Gray B. M., 1979, J. Immun. Methods, 28:187-192).
There are some cross-reactions among serotypes; for example: serotypes 3, 6 and 8, serotypes 1, 9 and 11, and serotypes 7 and 4. It is not possible, so far, to differentiate these serotypes serologically. Most of these cross-reactions are due to the presence of common epitopes at the lipopolysaccharide (LPS) level. In addition, other cross-reactions, which are not found in serotyping, could be observed in serological analysis of chronically infected animals that are continuously challenged with the microorganism. These cross-reactions are usually associated with outer membrane proteins (cell wall proteins, iron-repressible proteins, etc.) and rough lipopolysaccharides. However, it is important to remember that one herd, and even one animal, might be infected with several serotypes simultaneously. In this case, the detected antibodies against different serotypes are probably not cross-reactions, but homologous and specific reactions. This is one of the most important problem to be solved by the use of specific and sensitive serological tests in accordance with the present invention.
Healthy carrier pigs may be responsible for the transmission of the disease. The absence of clinical signs and/or lesions at the slaughter-house does not implicate necessarily the absence of the infection.
Following infection, antibodies can usually be detected in 10-15 days. Some animals will remain serologically positive for a few months, but most will be positive for a long period of time; once more, it will depend on the test used.
The proportion of seropositive sows as well as their titers tended to decrease with age.
Isolation of A. pleuropneumoniae from apparently healthy carrier pigs is difficult; it probably should be used as a complement to the serology in conflictive cases.
The development of better serological tests is a necessity because the infection still has an economic impact on the swine industry and the current vaccines are not effective.
To date, there exist no stable kit for the effective serodiagnosis of pig pleuropneumonia in the field.
It would be highly desirable to be provided with a kit for readily determining the presence of antibodies against A. pleuropneumoniae serotype 2 in a serum sample.
It would be highly desirable to be provided with such ELISA diagnostic kit for A. pleuropneumoniae which could be used for A. pleuropneumoniae serodiagnosis while remaining in the field.
One aim of the present invention is to provide a kit for the accurate, rapid and sensitive assay of antibodies against A. pleuropneumoniae serotype 2 in a sample.
Another aim of the present invention is to provide an ELISA diagnostic kit for A. pleuropneumoniae to be used for A. pleuropneumoniae serodiagnosis while remaining in the field. The novelty and originality of the ELISA diagnostic kit of the present invention resides in the particular combination of a novel purification method of the antigen to be used and a novel sensitization and stabilization methods of the plates of the kit.
The kits of the present invention differs from the ELISA method of the prior art for the determination of A. pleuropneumoniae antibodies. In the prior art method, the antigen is fixed to the plates in a PBS buffer and the plates are immediately used after the antigen fixation is completed. The prior art method may include a computerized reading protocol for the determination of the antibodies in the samples as described by Trottier, Y. L. et al. (1992, J. Clin. Microbiol., 30:46-53). The kits of the present invention mainly differ in that the antigen is purified using a higher concentration of phenol,the antigen fixation procedure is different, and the fixed antigen is stored in a horseradish peroxidase (HRP) conjugate stabilizing solution (sold by Calbiochem-Novabiochem Corporation, La Jolla, Calif. 92039, U.S.A., catalogue #516534, containing phosphate buffered saline pH 7.2, BSA, 0.1% thimerosal and caprylic acid).
Thus, the purified antigen is resuspended in a PBS-EDTA buffer which is then added to each well of the plate. After an 18 hour incubation, horseradish peroxidase (HRP) conjugate stabilizing solution is added to each well. The antibodies in the samples can be determined visually by adding a chromogen, preferably 3,3xe2x80x2,5,5xe2x80x2-tetramethylbenzidine, e.g. TMBlue(trademark) (Biovest Inc., Massachusetts, USA)(see U.S. Pat. No. 5,013,646, issued May 7, 1991). The kits of the present invention, when compared to the prior art ELISA method, demonstrate a relative sensitivity and a relative specificity of 100%.
In accordance with another embodiment of the present invention there is provided an ELISA diagnostic kit for the assay of A. pleuropneumoniae serotype 2 antibodies in the serum of pigs comprising in separate packaging, at least one of the following:
a) a plate or solid support having bound thereto a purified lipopolysaccharide A. pleuropneumoniae serotype 2 antigen for a specific binding to anti-A. pleuropneumoniae serotype 2 antibodies present in the serum of pigs;
b) serum from pigs experimentally inoculated with a strain of A. pleuropneumoniae serotype 2 to serve as a positive control;
c) pig serum from a specific pathogen free herd to serve as a negative control; and
d) a detectably labeled conjugate which binds to the pig antibodies bound to the plate of a).
The antigen of step a) when bound to a solid support can be stabilized by storing it at 4xc2x0 C. in the HRP conjugate stabilizing solution which keeps the bound antigen stable for at least 25 weeks.
The ELISA diagnostic kits of the present invention may further comprise the following:
e) a substrate which allows the visualization of the detectably labeled conjugate.
In accordance with another embodiment of the present invention there is provided a method for the preparation of the kit, which comprises the steps of:
a) purifying lipopolysaccharide A. pleuropneumoniae serotype 2 antigen by phenol extraction and centrifugation of said antigen bacterial crude extract;
b) fixing the antigen of step a) to a solid support and stabilizing said fixed antigen;
c) immunizing mammals with a strain of A. pleuropneumoniae serotype 2 and collecting serum to serve as positive control sera; and
d) collecting sera from A. pleuropneumoniae free herds to serve as negative control sera.
The stabilizing of the solution of step b)may be achieved by storing it in an HRP conjugate stabilizing solution at 4xc2x0 C., which keeps the bound antigen stable for at least 25 weeks. In a particular embodiment, the HRP conjugate stabilizing solution contains phosphate buffered saline pH 7.2, 20 mg/ml BSA, 0.1% thimerosal and a saturating amount of caprylic acid.